Aim: The present study was designed to investigate the effect of thymoquinone (TQ) on the levels of glycoprotein components in plasma and tissues of streptozotocin (STZ)-nicotinamide (NA)-induced diabetic rats. Materials and Methods: Diabetes was induced in experimental rats by a single intraperitoneal (i.p) injection of STZ (45 mg/kg b.w) dissolved in 0.1 M citrate buffer (pH 4.5) 15 minutes after the i.p injection of NA (110 mg/kg b.w). Results: The levels of hexose, hexosamine, fucose, and sialic acid were estimated in plasma, liver, and kidney tissues of experimental rats. An increase in glycoprotein components in plasma was noticed in diabetic rats. In hepatic and renal tissues, a significant decrease in sialic acid with increases in hexose, hexosamine, and fucose levels were observed in diabetic rats when compared with control animals. Oral administration of TQ at 80 mg/kg b.w. for 45 days significantly ameliorated the glycoprotein changes in plasma and tissues of diabetic rats. Conclusion: The results of the present study show the potent beneficial effects of TQ in modifying the levels of glycoprotein components in plasma and tissues of diabetic rats.

Diabetes mellitus is a heterogeneous syndrome characterized by hyperglycemia resulting from impaired insulin secretion, action, or both. [1] According to World Health Organization, the number of patients with type 2 diabetes mellitus (T2DM) will be more than 300 million by 2030. [2] The debilitating aspect of diabetes is the numerous complications that affect the functions of various organs. [3] Though insulin resistance and beta-cell failure are considered as the central process in the development of diabetes mellitus, studies showed that impaired metabolism of glycoproteins play a vital role in the pathogenesis and in its complications. [4]

Glycoproteins are widely distributed proteins that contain one or more covalently linked carbohydrate chains. They function as hormones, enzymes, blood group antigens, and constituents of extracellular membranes. [5] The principle types of sugar present are hexose, hexosamine, fucose, and sialic acid, which encode the diverse biological functions of glycoproteins. Protein-bound hexose imparts hydrophilic nature to the cell membrane, and hexosamine through its cationic charges makes the cell membrane more polar. [6] Sialic acid is an abundant terminal monosaccharide of glycoconjugates. About 70% of total sialic acid is found on the cell surface, where it plays a pivotal role in cell-to-cell and cell to matrix interactions. [7] L-fucose, a deoxyhexose is a component of many N- and O-linked glycoproteins and participates in biological recognition events. [8]

Under physiological conditions, the levels of serum and tissue glycoproteins are maintained in a narrow range. Profound alterations in the metabolism of glycoproteins are noticed in diabetes and in cardiovascular diseases. [9] Such changes are also implicated in the pathogenesis of liver and kidney diseases in diabetes mellitus. [10] Aberrations in glycoprotein metabolism are observed in naturally occurring and in experimental diabetes. [11]

Though insulin and oral hypoglycemic agents are available to manage diabetes, still it remains a challenge in preventing the onset of diabetic complications. [12] Therefore, there is an imperative need for novel therapeutic agents that can overcome the demerits of existing therapeutic modalities. Plants are an exemplary source of drugs. A wide range of plant-derived active principles are commonly used in the treatment of diabetes, which are easily available, economical, and are free from adverse side effects. Nigella sativa, a member of Ranunculaceae family of flowering plants, is extensively investigated for its nutritive and therapeutic purposes. It is an annual herb incorporated in diets and everyday lifestyles to promote health and to treat diseases. Thymoquinone (TQ), the main constituent of the essential oil of Nigella sativa seeds, is an active quinone which possesses diverse pharmacological activities such as anti-inflammatory. [13] antidiabetic, [14] and antioxidant activities. [15]

To date, no such information exists to explain the effect of TQ on the components of glycoprotein in diabetic rats. Therefore, the present study was designed to assess the role of TQ on the levels of plasma and tissue glycoprotein components in streptozotocin (STZ)-nicotinamide (NA)-induced diabetic rats. Recently, we reported that TQ at 80 mg/kg b.w. exhibited significant antidiabetic effect. [14] Therefore, the same dose was fixed in the present study.

Materials and Methods

0Animals

Male albino rats of Wistar strain (200-220 g) were procured from Central Animal House, Rajah Muthiah Medical College (RMMC), Annamalai University. They were acclimatized to animal house conditions, and fed with standard pellet diet (Hindustan Lever Limited, Mumbai, India) and water ad libitum. The rats were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research Hyderabad, India and were approved by Institutional Animal Ethical Committee (Vide. No: 564, 2008), Annamalai University.

Drug and chemicals

TQ and STZ were purchased from Sigma Chemical Co (St. Louis, MO, USA) and NA was purchased from Ranbaxy Chemicals Ltd. All the other chemicals used were of analytical grade.

In this experiment, a total of 24 rats (12 diabetic surviving rats and 12 normal rats) were used. The rats were divided into four groups of six rats each. Group 1: Normal control rats (vehicle treated), Group 2: Normal rats intragastrically received TQ (80 mg/kg b.w.) dissolved in 1 mL of corn oil for 45 days, Group 3: Diabetic control rats, Group 4: Diabetic rats intragastrically administered with TQ (80 mg/kg b.w.) dissolved in 1 mL of corn oil for 45 days.

At the end of the experimental period, animals were fasted overnight, anesthetized using ketamine (24 mg/kg b.w., intramuscular injection), and sacrificed by cervical decapitation. All biochemical studies are carried out on plasma, liver, and kidney tissues of control and experimental rats. Plasma proteins are precipitated by adding 95% ethanol and the precipitate was used for the estimation of protein-bound hexose and hexosamine.

Determination of glycoprotein levels in plasma and tissues

For the estimation of glycoproteins, the tissues were defatted by the method of Folch et al [17] and hydrolyzed with 0.1N H2SO4 at 80°C for 1 hour, and aliquots were used for sialic acid estimation by the method of Warren. [18] To the remaining solution, 0.1 N NaOH was added and aliquots were used for the estimation of hexose and hexosamine and fucose by the methods of Niebes [19] Wagner, [20] and Dische and Shettles [21] respectively.

Statistical analysis

The data from various biochemical parameters were analyzed using analysis of variance (ANOVA) and the group means were compared by Duncan's Multiple Range Test using a statistical software package (SPSS for Windows, V.13.0, Chicago, IL, USA). Results were presented as mean ± S.D. and P<0.05 were considered as statistically significant and included in the study. [22]

Results

Effect of TQ on plasma glycoproteins

[Table 1] shows the changes in the levels of protein-bound hexose, hexosamine, fucose, and sialic acid in plasma of control and experimental rats. Significantly higher levels of glycoprotein components were observed in the plasma of diabetic rats when compared with normal control rats. Administration of TQ resulted in a significant reduction of glycoprotein components in plasma of diabetic rats when compared with diabetic control rats.

Table 1: Effect of TQ on the levels of plasma glycoprotein components in control and experimental rats

The levels of protein-bound hexose, hexosamine, fucose, and sialic acid in liver and kidney tissues of control and experimental rats are shown in [Figure 1], [Figure 2], [Figure 3] and [Figure 4]. The levels of sialic acid was significantly decreased in the tissues of diabetic rats, whereas increase in protein-bound hexose, hexosamine, and fucose were observed. Oral administration of TQ to diabetic rats significantly reversed these changes in tissues to near normal.

Figure 1: Effect of TQ on protein-bound hexose in liver and kidney tissues of control and experimental rats. Values are given as mean ± S.D for six rats in each group. Values not sharing a common superscriptletter differ signifi cantly at P<0.05 (DMRT)

Figure 2: Effect of TQ on protein bound hexosamine in liver and kidney tissues of control and experimental rats. Values are given as mean ± S.D for six rats in each group. Values not sharing a common superscript letter differ signifi cantly at P<0.05 (DMRT)

Figure 3: Changes in the levels of sialic acid content in liver and kidney tissues of control and experimental rats. Values are given as mean ± S.D for six rats in each group. Values not sharing a common superscript letter differ signifi cantly at P<0.05 (DMRT)

Figure 4: Changes in fucose content of liver and kidney tissues of control and experimental rats. Values are given as mean ± S.D for six rats in each group. Values not sharing a common superscript letter differ signifi cantly at P<0.05 (DMRT)

Diabetes is a progressive disease whose biochemical basis for progression and complications are poorly understood. In the diabetic state, glucose is redirected through insulin-independent pathways resulting in enhanced production of carbohydrate moieties of glycoproteins. Therefore, there is an elevation in the levels of protein-bound hexose, hexosamine, fucose, and sialic acid in plasma of diabetic animals.

Several molecular mechanisms are implicated in hyperglycemia-induced metabolic disturbances in diabetes. They include increased flux through polyol pathway, formation of advanced glycation end products, activation of PKC isoforms, and increased activity of hexosamine pathway. Hexosamine biosynthetic pathway represents a minor metabolic route of glucose at fructose 6-phosphate step of glycolysis. This pathway is considered as a sensor of nutrients and an increase in this pathway is regarded as a key factor in the metabolic complications of diabetes. [23] Sustained hyperglycemia due to insulin deficiency with concomitant oxidative stress increases the expression of GFAT (Glutamine: Fructose 6-phosphate amino transferase), the rate-limiting enzyme of this pathway leading to an increase in the levels of hexosamine. [24]

Liver is the major site of glycoprotein synthesis whose functions are drastically altered in diabetes. The activities of fucosidase and fucosyltransferases are increased in STZ-induced diabetic rats. [25] There occurs an eight-fold increase in serum fucose levels in diabetics than in normal individuals. Fucosylation reactions confer unique functional properties to glycoproteins. An elevation of fucosylated proteins in diabetic rats could be due to an increase in the synthesis or degradation of these proteins.

An elevation in serum sialic acid levels is observed in T2DM and is also a risk factor for microvascular complications. Oxidative stress and inflammation brings damages to cellular membranes and increases serum sialic acid levels. In addition, vascular endothelium is rich in sialic acid moieties where it regulates permeability. Impaired function of insulin and the resulting hyperglycemia are associated with endothelial dysfunction, leading to the release of sialic acid into circulation. [26] Thus in diabetes, there is a consistent increase in plasma sialic acid levels, whereas its content varies in different tissues. [27] The diminished activity of enzymes of sialic acid biosynthesis explains the decreased sialic acid content in liver of diabetic experimental animals. This decrease may also be related to increased synthesis of fibronectin which contains sialic acid in its core structure.

The altered levels of plasma and tissue glycoprotein components observed in the present study are in accordance with studies on experimental diabetic rats.[28] We observed a significant improvement in plasma and tissue glycoprotein components in diabetic rats treated with TQ. This is attributed to the insulinotropic action of TQ which restored the disturbed glycoprotein components in plasma and tissues of diabetic animals to near normal. This shows the efficacy of TQ in modulating the altered glycoprotein metabolism in diabetic animals.

Conclusion

The present finding shows the beneficial effect of TQ in ameliorating the disturbed glycoprotein components in diabetic rats. This can be used as an effective indicator to demonstrate its effects in controlling the complications of diabetes. The effects of TQ on the activities of enzymes of glycoprotein metabolism are yet to be elucidated.